Role Of AIM2-inflammasome In The Pathogenesis Of Rheumatoid Arthritis And Effect Of Sanshui Baihu Decoction On AIM2-inflammasome Pathway

BackgroudRheumatic arthritis(RA)is a chronic immune mediated inflammatory disease that is characterised by haperproliferation of synovial cells,infiltration of mononuclear cells into the synoium and early destruction of articular cartilage and bone,causing progresive damage to the muscluoskeletal symstem and consequently loss of physical ablity and life quality.Therapies for RA,ranging from NSAIDs to DMRADS and biological DMARDS,still cause severe side effect and are only induced remission in around 20-30%of the patients,leaving the majority of the individual affected by RA with a chronic inflammatory process that will lead to damage.The role of IL-1β and IL-18 in the pathophysiology of RA has been well established.The processing of bioactive IL-1β and IL-18 depends on activition of caspase-1 in protein complex termed inflammasome.The most intensely studied example of this is the inflammasome formed by the NLR family member NLRP3,but AIM2 inflamamsome,in which ASC acts as an adaptor protein for the activation of caspase-1,has not been well investigated.Therefore,a deeper understanding of AIM2 inflammasome offers a prospect of new therapeutic strategies for RA.In this study,we aim to establish the role of AIM2 inflammasome in the pathogenesis of RA.ContentChapter 1 Expression of differences of AIM2 inflammasome pathway related proteins in RA and OAObjectiveTo observe the expression of level of AIM2 between RA and OA patients in sera as well as synovium.To analyze the correlation between the expression of AIM2 inflammasome pathway related proteins and clinical inflammatory biomarkers including ESR,CRP.MethodSera of 49 case RA patients and 25 case OA patients were enrolled in ELISA assay.Synovial tissue samples were collected from 41 and 26 cases of RA and OA,respectively.Horseradish peroxidase immunohistochemical staining was used to detect AIM2 inflammasome pathway-related proteins,including:absent in melanoma 2(AIM2),apoptosis-associated speck-like protein containing a CARD(ASC),CASPASE-1,and interleukin-1(IL-1 β).A semi-quantitative score(H-score)was performed according to the degree of positiveness.Correlation analysis between H-score results and clinical indicators of ESR and C-reactive protein(CRP)were performed.ResultsThe H scores of AIM2 protein in RA synovial tissues was 131.9±7.25 and 54.37±7.52 in OA synovial tissues(t=7.42,P<0.001);The H scores of ASC protein in RA synovial tissues was 106.9±9.45 and 74.09±6.28 in OA synovial tissues(t=2.36,P<0.05);The H scores of CASPASE-1 protein in RA synovial tissues was 99.29±5.27 and 73.81 ± 10.09 in OA synovial tissues(t=2.15,P<0.05);The H scores of IL-1β protein in RA synovial tissues was 117.6±10.96 and 76±7.01 in OA synovial tissues(t-3.30,P<0.05).In the correlation analysis,AIM2 was positively correlated with ESR(r=0.74,P<0.01,95%CI:0.38-0.9),and ASC was positively correlated with ESR(r=0.5,P<0.05,95%CI:0.16-0.74).),IL-1β was positively correlated with ESR(R=0.62,P<0.05,95%CI:0.31-0.81),and the difference was statistically significant(P<0.05).At the same time,AIM2 was positively correlated with C-reactive Protein(R=0.65,P<0.05,95%CI:0.25-0.86);ACS was positively correlated with C-reactive Protein(r=0.42,P<0.05,95%CI:0.05-0.69).IL-1β was positively correlated with C-reactive Protein(r=0.41,P<0.05,95%CI:0.05-0.67)and positively correlated with C-reactive protein,and the difference was statistically significant(P<0.05).ConclusionThe expression of AIM2 inflammasome pathway-related proteins in RA synovium,including AIM2,ASC,CASPASE-1,and IL-1β,is higher than that of OA and are positively correlated with activity;Activation of AIM2 inflammasome pathway may be RA activity mechanisms.Chapter 2 Different expression of differences of AIM2 in RA and OA and role of AIM2 inRASFsObjectiveTo determine the level of expression of AIM2 in RA and OA fibroblast-like synoviocytes and to investigate the role of AIM2 in RASFs.MethodImmunofluorecent staining was used to determined the intensity of AIM2 in RA and OA synovium.SiRNA AIM2 was transferred to RASFs and observe its effects on proliferation and migration by CCK-8 assay and transwell test respectively.CCK-8 assay,flow cytometry and transwell test were used to evaluate the proliferation,apoptosis and migration of RASFs.ResultExpression of AM2 was higher in RASFs than in OA fibroblast-like synoviocytes.After transferred AIM2 siRNA to FLS and incubation for 48 hours,the proliferation of FLS were significantly inhibited,and the apoptosis rate were significantly increased compared to FLS in control group.However,no effect on migration was detected.ConclusionAIM2 participated in the proliferation of FLS,and might be a potential target for therapy.Chapter 3 Effect of SSBH decotion on AIM2 inflammasome pathwayObjectiveTo investigate the effect of SSBH decotion on AIM2 inflammasome pathway.MethodSSBH containing serum and control serum was obtained from blood sample of SD rats by gavage for a week.In vitro study was divided into 3 groups:10%NS group,10%SSBH group and 20%SSBH group.After treated with SSBH serum and contrl serum,proliferation and migration properties of RASFs were determined by CCK-8 assay and wound healing assay.Meanwhile,the mRNA and protein level of AIM2 inflammasome pathway related protein including AIM2,ASC,Caspase-1 and IL-1βwere evaluated by RT-PCR and western blot,respectively.ELISA was used to determine the level of IL-1β in supernatant after incubation for 48h.ResultCompared with control group,10%SSBH and 20%SSBH significantly inhibited the proliferation of synovial fibroblasts after 48 and 72 hours incubation(P<0.05);10%SSBH and 20%SSBH inhibited synovial fibroblasts.The average healing rate of the NS group was 52 ± 0.02%,and the average healing rates of the 10%SSBH and 20%SSBH were 28±0.01%,2±0.07%respectively,which were lower than that of the control group.The difference was statistically significant.(P<0.05).SSBHT down-regulated the mRNA and protein expression of Aim2-inflammasome signaling pathway related proteins compared to the control group.IL-1β in the 20%SSBH group was significantly reduced and the difference was statistically significant(P<0.05)ConclusionSSBH decotion inhibited the proliferation and migration of synovial fibroblasts via down-regulation of Aim2-inflammasome signaling pathway.Conclusion(1)AIM2 inflammasome signaling pathway may be involved in the pathogenesis of RA(2)The high expression of AIM2 in RA synovial fibroblasts promotes their proliferation.(3)SSBH may play an anti-RA role by down-regulating the AIM2 pathway.